CN109799220B - Method for detecting histamine in tissue fluid based on metal chelate Raman label technology - Google Patents
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Abstract
The invention discloses a method for detecting histamine in tissue fluid based on a metal chelate Raman label technology, which comprises the following steps: taking an organic ligand and divalent nickel salt to carry out coordination reaction to obtain a metal chelate solution; coating the metal chelate solution on the surface of the surface enhanced Raman substrate with negative charges on the surface, and drying to obtain a functionalized substrate; mixing alcohol and tissue fluid, centrifuging, and collecting supernatant; adding an acidic solution into the supernatant, uniformly mixing, adding an organic solvent, uniformly mixing, centrifuging, and taking a water-phase solution; and coating the aqueous phase solution on a functionalized substrate, and detecting by using a Raman spectrometer. The method for detecting histamine in tissue fluid based on the metal chelate Raman label technology provided by the invention has the advantages of simple steps and good SERS detection sensitivity, stability and repeatability for histamine.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a method for detecting histamine in tissue fluid based on a metal chelate Raman label technology.
Background
Histamine is one of the self-active substances, and is mainly stored in mast cells and basophils in the human body. When a human body is stimulated by the allergen, histamine is released to cause vasodilatation, so that edema and blood pressure are reduced, various corresponding symptoms are generated, and the health of the human body is damaged. As an inflammatory mediator of immediate hypersensitivity, anaphylactic shock and even death occurs when histamine in the blood reaches a certain level. Histamine is a good target molecule for drug treatment of allergic diseases and is an auxiliary index for definite diagnosis of allergic diseases. Therefore, the development of a fast, simple, specific and sensitive histamine detection method is an urgent need for the early diagnosis or prevention of allergic reactions.
In 1974, Fleischmann et al obtained high-quality raman spectra of pyridine molecules adsorbed on the surface of a roughened silver electrode when the surface of the silver electrode was electrochemically roughened and characterized by a raman spectrometer. In 1977, VanDuyne et al analyzed this phenomenon to indicate that it is an enhancement effect with some inevitable relationship to the roughened Surface, which effect was later referred to as the Surface-enhanced Raman Scattering (SERS) effect. Since SERS finds itself, it is often used as a spectrum tool for sensitive molecular fingerprint identification due to its advantages of high sensitivity, no damage, fingerprint characteristics, and fast detection speed.
Because the common allergic reaction inflammatory mediator detection method generally has high detection cost and long time consumption and needs professional personnel, the detection of the SERS technology can make up the defects of the method. Small molecule markers such as histamine are usually present in very low amounts in blood or interstitial fluid, and also contain a large number of other biological interfering components. Meanwhile, the Raman scattering cross section is small, and the actual detection requirement is difficult to meet by directly using SERS detection.
Disclosure of Invention
Based on the technical problems in the background art, the invention provides a method for detecting histamine in tissue fluid based on a metal chelate Raman label technology, which has the advantages of simple steps and good SERS detection sensitivity, stability and repeatability for histamine.
The invention provides a method for detecting histamine in tissue fluid based on a metal chelate Raman label technology, which comprises the following steps:
s1, taking an organic ligand to perform a coordination reaction with a divalent nickel salt to obtain a metal chelate solution;
s2, coating the metal chelate solution on the surface of the surface enhanced Raman substrate with negative charges on the surface, and drying to obtain a functionalized substrate;
s3, uniformly mixing alcohol mutually soluble with water and interstitial fluid, centrifuging, and taking supernatant;
s4, adding an acidic solution into the supernatant in the S3, uniformly mixing, adding an organic solvent, uniformly mixing, centrifuging, and taking an aqueous phase solution;
s5, coating the aqueous phase solution in the S4 on the functionalized substrate in the S2, and detecting by using a Raman spectrometer.
Preferably, the specific steps of S1 are: mixing the organic ligand with a divalent nickel salt solution, adjusting the pH value to 5-7, and stirring to obtain a metal chelate solution.
Preferably, in S1, the organic ligand is a monodentate organic ligand or a polydentate organic ligand.
Preferably, the organic ligand is one of citric acid, ethylenediamine tetraacetic acid, nitrilotriacetic acid and pyridine.
Preferably, in S1, the divalent nickel salt is a divalent nickel salt dissolved in water.
Preferably, the divalent nickel salt is one or a mixture of nickel nitrate hexahydrate, nickel acetate and nickel chloride.
Preferably, in S2, the metal chelate solution is dropped on the surface of the surface-enhanced raman substrate with a negative surface charge, and the functionalized substrate is obtained after drying.
Preferably, the surface-enhanced raman substrate with a negatively charged surface is prepared according to the following process: soaking the silicon wafer in aqua regia for 2-3h, cleaning, soaking in piranha washing solution for 0.5-1.5h, cleaning with ultrapure water, and blow-drying with nitrogen to obtain clean silicon wafer; and centrifuging the gold nano sol with the average particle size of 50nm, dripping the gold nano sol on the surface of a clean silicon wafer, and drying to obtain the surface enhanced Raman substrate with negative charges on the surface.
Preferably, in S3, the tissue fluid is one of serum, muscle tissue fluid, synovial tissue fluid and epidermal tissue fluid.
Preferably, in S3, the volume ratio of water-miscible alcohol to interstitial fluid is 0.1-0.5: 0.1.
preferably, the water-miscible alcohol is one or more of ethanol, methanol and n-butanol.
Preferably, in S4, the volume ratio of the supernatant, the acidic solution and the organic solvent is 10: 0.1-1: 2-3.
Preferably, in S4, the acidic solution is one of a strong acid solution, a strong acid and weak base salt solution, and a weak acid solution; the organic solvent is an organic solvent which has a density lower than that of water and is immiscible with water.
Preferably, the acid solution is one or a mixture of hydrochloric acid, sulfuric acid, acetic acid and nitric acid; the organic solvent is one or a mixture of more of petroleum ether, cyclohexane, diethyl ether and dimethyl ether.
Preferably, the organic solvent is petroleum ether.
Preferably, in S5, the excitation light wavelength of the raman spectrometer is 633 nm.
Preferably, the serum is obtained by taking supernatant after blood standing coagulation.
Preferably, the piranha washing liquor is prepared from concentrated sulfuric acid and hydrogen peroxide according to a volume ratio of 3: 1 is configured.
Preferably, the gold nanosol is prepared by taking one or more of citric acid, ascorbic acid and sodium borohydride as a reducing agent.
The histamine molecule can be combined with metal ions to form a chelate, forming a raman active substance with significant absorption in the ultraviolet visible region. Resonance raman scattering occurs when the energy of the incident or scattered light matches the energy of a certain electron transition of the chelate. When Resonance Raman Scattering occurs, the frequency of the Raman transition is greatly increased, giving sufficient Raman spectral intensity even at the single molecule level to be detected, i.e. Surface-enhanced Resonance Raman spectroscopy (SERRS). The method for detecting histamine in tissue fluid based on metal chelate Raman label technology realizes the rapid capture and sensitive detection and analysis of histamine in tissue fluid by using the strategy of metal chelate Raman label through the resonance enhancement principle.
In the method for detecting histamine in tissue fluid based on metal chelate Raman label technology, the histamine in the tissue fluid such as serum, muscle tissue fluid, synovial tissue fluid, epidermal tissue fluid and the like after SERS detection treatment is analyzed according to the scientific principle as follows: firstly, divalent nickel ions are combined with a monodentate or polydentate organic ligand to prepare a metal chelate, and the chelate is used as a substance for selectively capturing histamine by using a Raman label; then preparing an enhanced Raman substrate with negative charges on the surface, coating the metal chelate on the surface of the substrate, adsorbing a metal chelate Raman label through hydrogen bonds and chemical action, and simultaneously regulating and controlling the assembly of the enhanced Raman substrate, thereby generating uniform and high-activity SERS 'hot spots'; histamine is in the body of a living organism, and the environment of the organism is complex. The metal chelate is used as a Raman label and is chelated and coordinated with histamine, and the probability of resonance transition under laser irradiation is greatly increased, so that resonance enhancement is generated, and the detection sensitivity is improved. Because the tissue fluid contains a large amount of protein or a plurality of fat-soluble micromolecules which can interfere the detection of histamine, a rapid pretreatment method aiming at the histamine is established, the tissue fluid is treated by sequentially adopting alcohol, acid solution and organic solvent, wherein the characteristic that the solubility of the protein in water can be obviously reduced by using the organic solvent which is mutually soluble with water is utilized, the aim of separating and purifying the protein is realized by adding alcohol, and in addition, the acid-base property of the body fluid is adjusted by adding the acid solution because the histamine is more stable under the condition of medium acidity. Finally, the selected organic solvent is an organic solvent which has density less than that of water and is immiscible with water, and the organic solvent required by protein purification can be separated by centrifugation, and the corresponding water phase positioned at the lower layer is taken after centrifugation to achieve the purposes of separation and purification.
The invention has the following beneficial effects:
1. the invention separates and enriches histamine in the tissue fluid, thereby avoiding the interference of other impurities in the tissue fluid on the detection of target molecules; secondly, a functional SERS substrate is constructed by designing a metal chelate Raman label, so that the detection sensitivity is improved; the functionalized substrate is constructed, so that the detection sensitivity, stability and repeatability of histamine in tissue fluid can be further improved.
2. The enhanced substrate is negatively charged and can be combined with the metal chelate Raman label through chemical action and hydrogen bonds; secondly, the assembled substrate is uniform and arranged in a single layer and has a large number of 'hot spots', so that the sensitivity and the repeatability of detection are improved.
3. The tissue fluid of the invention has various types, including serum, muscle tissue, synovial tissue and epidermal tissue, can meet the requirement of detecting material difference in practical detection, and has wide application range.
4. The histamine can be separated and purified by adding three reagents into the tissue fluid, and the influence of other impurities (saccharides, amino acids, proteins, phospholipids) can be avoided.
5. The purification process of the invention is fast, the treatment process of the tissue fluid can be completed within about 5 to 10 minutes, and the process is convenient and fast.
6. The invention has higher sensitivity, selectivity, repeatability and stability to histamine in tissue fluid, and the detection limit is below 1 mu M.
Drawings
FIG. 1 is a SERS detection spectrum of serum containing Histamine (HA) and serum;
FIG. 2 is a graph of SERS spectra of sera of examples 1-4 with various concentrations of histamine added;
FIG. 3 is a SERS detection spectrum of muscle tissue fluid and muscle tissue fluid containing histamine in example 5;
FIG. 4 is a SERS detection spectrum of synovial fluid containing histamine and synovial fluid in example 6.
Detailed Description
The technical solution of the present invention will be described in detail below with reference to specific examples.
Example 1
The invention provides a method for detecting histamine in tissue fluid based on a metal chelate Raman label technology, which comprises the following steps: soaking the silicon wafer in aqua regia for 2h, cleaning, soaking in piranha washing solution for 1h, cleaning with ultrapure water, and drying with nitrogen to obtain a clean silicon wafer; centrifuging gold nano sol with the average particle size of 50nm, then dropwise adding the gold nano sol on the surface of a clean silicon wafer, and drying to obtain a surface enhanced Raman substrate with negative charges on the surface for later use; mixing nitrilotriacetic acid and nickel nitrate hexahydrate solution, adjusting the pH value to 5, stirring to change the color of the solution from colorless to bluish purple to obtain metal chelate solution, dropwise adding the metal chelate solution to the surface of a surface enhanced Raman substrate with negative charges on the surface, combining a metal chelate Raman label with the surface enhanced Raman substrate through chemical action force, and drying to obtain a functionalized substrate; preparing a histamine solution with the concentration of 5 mu mol/L, taking 0.15mL, and adding 0.15mL of serum to obtain the serum containing histamine; taking 0.3mL of serum containing histamine into a 1.5mL centrifuge tube, adding 0.9mL of methanol, shaking up, centrifuging for 3min, taking 1mL of supernatant into another clean centrifuge tube, adding 30 μ L of hydrochloric acid, mixing uniformly, adding 0.2mL of organic solvent petroleum ether, oscillating, centrifuging for 2min, and taking an aqueous phase solution; and (3) dropwise adding 10 mu L of aqueous phase solution onto the functionalized substrate, detecting the aqueous phase solution by using a Raman spectrometer, recording the Raman intensity, and repeatedly testing for eight times.
Example 2
The only difference from example 1 is that: a histamine solution was prepared at a concentration of 1. mu. mol/L.
Example 3
The only difference from example 1 is that: a histamine solution was prepared at a concentration of 0.5. mu. mol/L.
Example 4
The only difference from example 1 is that: a histamine solution was prepared at a concentration of 0.1. mu. mol/L.
Example 5
The invention provides a method for detecting histamine in tissue fluid based on a metal chelate Raman label technology, which comprises the following steps:
s1, mixing nitrilotriacetic acid with a nickel nitrate hexahydrate solution, adjusting the pH value to 7, and stirring to form a metal chelate solution;
s2, soaking the silicon wafer in aqua regia for 3h, cleaning, soaking in piranha washing solution for 1h, cleaning with ultrapure water, and blow-drying with nitrogen to obtain a clean silicon wafer; centrifuging gold nano sol with the average particle size of 50nm to remove redundant surfactant, then dropping the gold nano sol onto a clean silicon chip, and drying to obtain a surface enhanced Raman substrate; dripping a metal chelate solution on the surface of the surface enhanced Raman substrate, and naturally drying to obtain a functionalized substrate;
s3, preparing a histamine solution with the concentration of 5 mu mol/L, taking 0.15mL, and adding 0.15mL of muscle tissue fluid to obtain muscle tissue fluid containing histamine; taking 0.3mL of muscle tissue fluid containing histamine into a 1.5mL centrifuge tube, adding 900 μ L of methanol into the muscle tissue fluid, shaking, centrifuging for 3min, and taking 1mL of supernatant into another clean centrifuge tube;
s4, adding 80 mu L of acetic acid into the centrifuge tube containing the supernatant, uniformly mixing, adding 300 mu L of organic solvent ethyl ether, oscillating, centrifuging for 2min, and taking the aqueous phase solution;
s5, dripping 10 mu L of aqueous phase solution on the functionalized substrate in the S2, detecting the aqueous phase solution by using a Raman spectrometer, recording the Raman intensity, and repeating the test for eight times.
Example 6
The invention provides a method for detecting histamine in tissue fluid based on a metal chelate Raman label technology, which comprises the following steps:
s1, mixing nitrilotriacetic acid and nickel nitrate hexahydrate solution, adjusting the pH value to 6, and stirring to obtain metal chelate solution;
s2, soaking the silicon wafer in aqua regia for 2h, cleaning, soaking in piranha washing solution for 1h, cleaning with ultrapure water, and blow-drying with nitrogen to obtain a clean silicon wafer; centrifuging gold nano sol with the average particle size of 50nm to remove redundant surfactant, then dropping the gold nano sol onto a clean silicon chip, and drying to obtain a surface enhanced Raman substrate; dripping a metal chelate solution on the surface of the surface enhanced Raman substrate, and naturally drying to obtain a functionalized substrate;
s3, preparing a histamine solution with the concentration of 5 mu mol/L, taking 0.15mL, and adding 0.15mL of synovial tissue fluid to obtain a synovial tissue fluid containing histamine; taking 0.3mL of synovium tissue fluid containing histamine to a 1.5mL centrifuge tube, adding 0.9mL of methanol to the synovium tissue fluid, shaking up, centrifuging for 3min, and taking 1mL of supernatant to another clean centrifuge tube;
s4, adding 30 mu L of nitric acid into the centrifuge tube containing the supernatant, uniformly mixing, adding 200 mu L of organic solvent methyl ether, oscillating, centrifuging for 2min, and taking the aqueous phase solution;
s5, dripping 10 mu L of aqueous phase solution on the functionalized substrate prepared in the S2, detecting the aqueous phase solution by using a Raman spectrometer, and recording the Raman intensity.
Fig. 1 is a SERS detection spectrogram of serum and serum containing Histamine (HA), wherein the upper spectrogram a is the SERS detection spectrogram of serum containing histamine, and the lower spectrogram b is the SERS detection spectrogram of serum; as can be seen from FIG. 1, the detection method can successfully detect histamine in serum samples, and the Raman spectrum is 1266cm-1，1388cm-1And 1574cm-1Characteristic peaks appear.
FIG. 2 is a graph of SERS spectra of sera of examples 1-4 with various concentrations of histamine added; as can be seen from FIG. 2, the serum samples at different concentrations all detected the signal of the target, and the lowest detection added histamine solution with a concentration of 0.1. mu. mol/L.
FIG. 3 is a SERS detection spectrum of the muscle tissue fluid containing histamine and the muscle tissue fluid in example 5, wherein a spectrum a is the SERS detection spectrum of the muscle tissue fluid containing histamine in example 5, and b is the SERS detection spectrum of the muscle tissue fluid; FIG. 4 is the SERS detection spectra of the synovial tissue fluid containing histamine and the synovial tissue fluid in example 6, wherein spectrum 1 is the SERS detection spectrum of the synovial tissue fluid containing histamine in example 6, and spectrum 2 is the SERS detection spectrum of the synovial tissue fluid; as can be seen from FIGS. 3 and 4, the detection of histamine in muscle tissue and synovial tissue, which is 1266cm, can be successfully achieved using the established pretreatment technique and the complex probe described-1，1388cm-1And 1574cm-1All belong to the characteristic peaks of histamine.
Example 7
The invention provides a method for detecting histamine in tissue fluid based on a metal chelate Raman label technology, which comprises the following steps:
s1, taking an organic ligand to perform a coordination reaction with a divalent nickel salt to obtain a metal chelate solution;
s2, coating the metal chelate solution on the surface of the surface enhanced Raman substrate with negative charges on the surface, and drying to obtain a functionalized substrate;
s3, uniformly mixing alcohol mutually soluble with water and interstitial fluid, centrifuging, and taking supernatant;
s4, adding an acidic solution into the supernatant in the S3, uniformly mixing, adding an organic solvent, uniformly mixing, centrifuging, and taking an aqueous phase solution;
s5, coating the aqueous phase solution in the S4 on the functionalized substrate in the S2, and detecting by using a Raman spectrometer.
Example 8
The invention provides a method for detecting histamine in tissue fluid based on a metal chelate Raman label technology, which comprises the following steps:
s1, taking an organic ligand to perform a coordination reaction with a divalent nickel salt to obtain a metal chelate solution;
s2, coating the metal chelate solution on the surface of the surface enhanced Raman substrate with negative charges on the surface, and drying to obtain a functionalized substrate;
s3, uniformly mixing alcohol mutually soluble with water and interstitial fluid, centrifuging, and taking supernatant;
s4, adding an acidic solution into the supernatant in the S3, uniformly mixing, adding an organic solvent, uniformly mixing, centrifuging, and taking an aqueous phase solution;
s5, coating the aqueous phase solution in the S4 on a functionalized substrate in the S2, and detecting by using a Raman spectrometer;
the specific steps of S1 are as follows: mixing an organic ligand with a divalent nickel salt solution, adjusting the pH value to 7, and stirring to obtain a metal chelate solution;
in S1, the organic ligand is citric acid;
in S1, the divalent nickel salt is nickel nitrate hexahydrate;
in S2, dripping the metal chelate solution on the surface of the surface enhanced Raman substrate with negative charges on the surface, and drying to obtain a functionalized substrate; the surface enhanced Raman substrate with the surface with negative charges is prepared according to the following process: soaking the silicon wafer in aqua regia for 2h, cleaning, soaking in piranha washing solution for 1.5h, cleaning with ultrapure water, and drying with nitrogen to obtain a clean silicon wafer; centrifuging gold nano sol with the average particle size of 50nm, dripping the gold nano sol on the surface of a clean silicon wafer, and drying to obtain a surface enhanced Raman substrate with negative charges on the surface;
in S3, the interstitial fluid is serum;
in S3, the volume ratio of water-miscible alcohol to interstitial fluid is 0.1: 0.1;
the water-miscible alcohol is methanol;
in S4, the volume ratio of the supernatant, the acidic solution, and the organic solvent is 10: 0.1: 3;
in S4, the acidic solution is sulfuric acid; the organic solvent is cyclohexane;
in S5, the wavelength of the excitation light of the Raman spectrometer is 633 nm.
Example 9
The invention provides a method for detecting histamine in tissue fluid based on a metal chelate Raman label technology, which comprises the following steps:
s1, taking an organic ligand to perform a coordination reaction with a divalent nickel salt to obtain a metal chelate solution;
s2, coating the metal chelate solution on the surface of the surface enhanced Raman substrate with negative charges on the surface, and drying to obtain a functionalized substrate;
s3, uniformly mixing alcohol mutually soluble with water and interstitial fluid, centrifuging, and taking supernatant;
s4, adding an acidic solution into the supernatant in the S3, uniformly mixing, adding an organic solvent, uniformly mixing, centrifuging, and taking an aqueous phase solution;
s5, coating the aqueous phase solution in the S4 on a functionalized substrate in the S2, and detecting by using a Raman spectrometer;
the specific steps of S1 are as follows: mixing an organic ligand with a divalent nickel salt solution, adjusting the pH value to 5, and stirring to obtain a metal chelate solution;
in S1, the organic ligand is nitrilotriacetic acid;
in S1, the divalent nickel salt is nickel acetate;
in S2, dripping the metal chelate solution on the surface of the surface enhanced Raman substrate with negative charges on the surface, and drying to obtain a functionalized substrate; the surface enhanced Raman substrate with the surface with negative charges is prepared according to the following process: soaking the silicon wafer in aqua regia for 3h, cleaning, soaking in piranha washing solution for 0.5h, cleaning with ultrapure water, and drying with nitrogen to obtain a clean silicon wafer; centrifuging gold nano sol with the average particle size of 50nm, dripping the gold nano sol on the surface of a clean silicon wafer, and drying to obtain a surface enhanced Raman substrate with negative charges on the surface;
at S3, the interstitial fluid is muscle interstitial fluid;
in S3, the volume ratio of water-miscible alcohol to interstitial fluid is 0.5: 0.1;
the alcohol which is mutually soluble with water is ethanol;
in S4, the volume ratio of the supernatant, the acidic solution, and the organic solvent is 10: 1: 2;
in S4, the acidic solution is a strong acid weak base salt solution; the organic solvent is an organic solvent which has density lower than that of water and is immiscible with water; the organic solvent is petroleum ether;
in S5, the wavelength of the excitation light of the Raman spectrometer is 633 nm.
Example 10
The invention provides a method for detecting histamine in tissue fluid based on a metal chelate Raman label technology, which comprises the following steps:
s1, taking an organic ligand to perform a coordination reaction with a divalent nickel salt to obtain a metal chelate solution;
s2, coating the metal chelate solution on the surface of the surface enhanced Raman substrate with negative charges on the surface, and drying to obtain a functionalized substrate;
s3, uniformly mixing alcohol mutually soluble with water and interstitial fluid, centrifuging, and taking supernatant;
s4, adding an acidic solution into the supernatant in the S3, uniformly mixing, adding an organic solvent, uniformly mixing, centrifuging, and taking an aqueous phase solution;
s5, coating the aqueous phase solution in the S4 on a functionalized substrate in the S2, and detecting by using a Raman spectrometer;
the specific steps of S1 are as follows: mixing an organic ligand with a divalent nickel salt solution, adjusting the pH value to 6, and stirring to obtain a metal chelate solution;
in S1, the organic ligand is pyridine;
in S1, the divalent nickel salt is a mixture of nickel nitrate hexahydrate and nickel chloride;
in S2, dripping the metal chelate solution on the surface of the surface enhanced Raman substrate with negative charges on the surface, and drying to obtain a functionalized substrate; the surface enhanced Raman substrate with the surface with negative charges is prepared according to the following process: soaking the silicon wafer in aqua regia for 2.5h, cleaning, soaking in piranha washing solution for 0.8h, cleaning with ultrapure water, and blow-drying with nitrogen to obtain a clean silicon wafer; centrifuging gold nano sol with the average particle size of 50nm, dripping the gold nano sol on the surface of a clean silicon wafer, and drying to obtain a surface enhanced Raman substrate with negative charges on the surface;
at S3, the interstitial fluid is epidermal interstitial fluid;
in S3, the volume ratio of water-miscible alcohol to interstitial fluid is 0.35: 0.1;
the alcohol which is mutually soluble with water is a mixture of ethanol, methanol and n-butanol;
in S4, the volume ratio of the supernatant, the acidic solution, and the organic solvent is 10: 0.6: 2.4;
in S4, the acidic solution is a mixture of hydrochloric acid, sulfuric acid, and acetic acid; the organic solvent is a mixture of petroleum ether, cyclohexane, diethyl ether and methyl ether;
in S5, the wavelength of the excitation light of the Raman spectrometer is 633 nm.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (13)
1. A method for detecting histamine in tissue fluid based on a metal chelate Raman label technology is characterized by comprising the following steps:
s1, taking an organic ligand to perform a coordination reaction with a divalent nickel salt to obtain a metal chelate solution;
s2, coating the metal chelate solution on the surface of the surface enhanced Raman substrate with negative charges on the surface, and drying to obtain a functionalized substrate;
s3, uniformly mixing alcohol mutually soluble with water and interstitial fluid, centrifuging, and taking supernatant;
s4, adding an acidic solution into the supernatant in the S3, uniformly mixing, adding an organic solvent, uniformly mixing, centrifuging, and taking an aqueous phase solution;
s5, coating the aqueous phase solution in the S4 on a functionalized substrate in the S2, and detecting by using a Raman spectrometer;
wherein the organic ligand is one of citric acid, ethylenediamine tetraacetic acid, nitrilotriacetic acid and pyridine.
2. The method for detecting histamine in tissue fluid based on metal chelate Raman label technology according to claim 1, wherein the specific steps of S1 are as follows: mixing the organic ligand with a divalent nickel salt solution, adjusting the pH value to 5-7, and stirring to obtain a metal chelate solution.
3. The method for detecting histamine in tissue fluid based on metal chelate raman label technique according to claim 1 or 2, wherein in S1, the divalent nickel salt is a divalent nickel salt dissolved in water.
4. The method for detecting histamine in tissue fluid based on metal chelate Raman label technology according to claim 1 or 2, wherein in S1, the divalent nickel salt is one or more of nickel nitrate hexahydrate, nickel acetate and nickel chloride.
5. The method for detecting histamine in tissue fluid based on metal chelate Raman label technology according to claim 1 or 2, wherein in S2, the metal chelate solution is dropped on the surface of the surface enhanced Raman substrate with negative charges on the surface, and the functionalized substrate is obtained after drying; the surface enhanced Raman substrate with the surface with negative charges is prepared according to the following process: soaking the silicon wafer in aqua regia for 2-3h, cleaning, soaking in piranha washing solution for 0.5-1.5h, cleaning with ultrapure water, and blow-drying with nitrogen to obtain clean silicon wafer; and centrifuging the gold nano sol with the average particle size of 50nm, dripping the gold nano sol on the surface of a clean silicon wafer, and drying to obtain the surface enhanced Raman substrate with negative charges on the surface.
6. The method for detecting histamine based on metal chelate raman label technique according to claim 1 or 2, wherein in S3, the tissue fluid is one of serum, muscle tissue fluid, synovial tissue fluid and epidermal tissue fluid.
7. The method for detecting histamine in tissue fluid based on metal chelate raman label technique according to claim 1 or 2, wherein the volume ratio of water-miscible alcohol to tissue fluid in S3 is 0.1-0.5: 0.1.
8. the method for detecting histamine in tissue fluid based on metal chelate Raman label technology according to claim 7, wherein in S3, the water-miscible alcohol is one or more of ethanol, methanol and n-butanol.
9. The method for detecting histamine in tissue fluid based on metal chelate Raman label technology according to claim 1 or 2, wherein the volume ratio of the supernatant, the acidic solution and the organic solvent in S4 is 10: 0.1-1: 2-3.
10. The method for detecting histamine in tissue fluid based on metal chelate raman label technique according to claim 1 or 2, wherein in S4, the acidic solution is one of strong acid solution, strong acid and weak base salt solution, and weak acid solution; the organic solvent is an organic solvent which has a density lower than that of water and is immiscible with water.
11. The method for detecting histamine in tissue fluid based on metal chelate raman label technique according to claim 10, wherein in S4, the acidic solution is a mixture of one or more of hydrochloric acid, sulfuric acid, acetic acid, nitric acid; the organic solvent is one or a mixture of more of petroleum ether, cyclohexane, diethyl ether and dimethyl ether.
12. The method for detecting histamine in interstitial fluid based on metal chelate raman label technique according to claim 11, characterized in that said organic solvent is petroleum ether.
13. The method for detecting histamine in tissue fluid based on metal chelate Raman label technology according to claim 1 or 2, wherein the wavelength of the excitation light of the Raman spectrometer is 633nm in S5.
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